Normal hemostasis is the result of a complex balance between the processes of clot initiation and formation (blood coagulation) and clot dissolution (fibrinolysis). The complex interactions between blood cells, specific plasma proteins and the vascular surface, maintain the fluidity of blood unless injury and blood loss occur.
Blood coagulation is the culmination of a series of amplified reactions in which several specific zymogens of serine proteases in plasma are activated by limited proteolysis. Nemerson, Y. and Nossel, H. L., Ann. Rev. Med., 33:479 (1982). This series of reactions results in the formation of an insoluble fibrin matrix which is required for the stabilization of the primary hemostatic plug. The interaction and propagation of the activation reactions occurs through the extrinsic and intrinsic pathways of coagulation.
The serine protease, thrombin, is the primary mediator of thrombus formation. Thrombin acts directly to cause formation of insoluble fibrin from circulating fibrinogen. In addition, thrombin activates the zymogen factor XIII to the active transglutaminase factor XIIIa which acts to covalently stabilize the growing thrombus by crosslinking the fibrin strands. Lorand, L. and Konishi, K., Arch. Biochem. Biophys., 105:58 (1964). Beyond its direct role in the formation and stabilization of fibrin rich clots, the enzyme has profound bioregulatory effects on a number of cellular components within the vasculature and blood. Shuman, M. A., Ann. NY Acad. Sci., 405:349 (1986).
It is believed that thrombin is the most potent agonist of platelet activation, and it has been demonstrated to be the primary pathophysiologic-mediator of platelet-dependent arterial thrombus formation. Edit, J. F. et al., J. Clin. Invest., 84:18 (1989). Thrombin-mediated platelet activation leads to ligand-induced inter-platelet aggregation principally due to the bivalent interactions between adhesive ligands such as fibrinogen with the platelet integrin receptor glycoprotein IIb/IIIa which assume their active conformation following thrombin activation of the cell. Berndt, M. C. and Phillips, D. R., Platelets in Biology and Pathology, pp 43-74, Elsevier/North Holland Biomedical Press (Gordon, J. L. edit. 1981). Thrombin-activated platelets can more effectively support additional thrombin production through the assembly of new prothrombinase (factor Xa and Factor Va) and tenase (factor IXa and factor VIIIa) catalytic complexes on the membrane surface of intact activated platelets and platelet-derived microparticles, following thrombin-mediated activation of the non-enzymatic cofactors V and VIII, respectively. Tans, G. et al., Blood, 77:2641 (1991). This positive feedback process results in the local generation of high concentrations of thrombin within the vicinity of the thrombus which supports further thrombus growth and extension. Mann, K. G. et al., Blood, 76:1 (1990).
In contrast to its prothrombotic effects, thrombin has been shown to influence other aspects of hemostasis. These include its effect as an important physiological anticoagulant. The anticoagulant effect of thrombin is expressed following binding of thrombin to the endothelial cell membrane glycoprotein, thrombomodulin. This is thought to result in an alteration of the substrate specificity of thrombin thereby allowing it to recognize and proteolytically activate the circulating zymogen, protein C, to give activated protein C (aPC). Musci, G. et al., Biochemistry, 27:769 (1988). The activation of protein C by thrombin in the absence of thrombomodulin is poor.
Thrombin has also been shown to be a potent direct mitogen for a number of cell types, including cells of mesenchymal origin such as vascular smooth muscle cells. Chen, L. B. and Buchanan, J. M., Proc. Natl. Acad. Sci. USA, 72:131 (1975). The direct interaction of thrombin with vascular smooth muscle also results in vasoconstriction. Walz, D. A. et al., Proc. Soc. Expl. Biol. Med., 180:518 (1985). Thrombin acts as a direct secretagogue inducing the release of a number of bioactive substances from vascular endothelial cells including tissue plasminogen activator. Levin, E. G. et al., Thromb. Haemost., 56:115 (1986). In addition to these direct effects on vascular cells, the enzyme can indirectly elicit proliferation of vascular smooth muscle cells by the release of several potent growth factors (e.g. platelet-derived growth factor and epidermal growth factor) from platelet .alpha.-granules following thrombin-induced activation. Ross, R., N. Engl. J. Med., 314:408 (1986).
Many significant disease states are related to abnormal hemostasis. With respect to the coronary arterial vasculature, local thrombus formation due to the rupture of an established atherosclerotic plaque is the major cause of acute myocardial infarction and unstable angina. Moreover, treatment of an occlusive coronary thrombus by either thrombolytic therapy or percutaneous transluminal coronary angioplasty (PTCA) is often accompanied by an acute thrombotic reclosure of the affected vessel which requires immediate resolution. With respect to the venous vasculature, a high percentage of patients undergoing major surgery in the lower extremities or the abdominal area suffer from thrombus formation in this vascular bed which can result in reduced blood flow to the affected extremity and a predisposition to pulmonary embolism with high risk of mortality. Disseminated intravascular coagulopathy is commonly associated with septic shock, certain viral infections and cancer and is characterized by the rapid consumption of coagulation factors and disseminated vascular microthrombosis which may result in leukocyte activation, inflammation and organ failure.
Arterial thrombosis is a major clinical cause of morbidity and mortality. It is the primary cause of acute myocardial infarction which is one of the leading causes of death in the Western world. Arterial rethrombosis also remains one of the primary causes of failure following enzymatic or mechanical recanalization of occluded coronary vessels using thrombolytic agents or percutaneous transluminal coronary angioplasty (PTCA), respectively. Ross, A. M., Thrombosis in Cardiovascular Disorder, p. 327, W. B. Saunders Co. (Fuster, V. and Verstraete, M. edit. 1991); Califf, R. M. and Wilierson, J. T., Id. at p 389. In contrast to thrombotic events in the venous vasculature, arterial thrombosis is the result of a complex interaction between fibrin formation resulting from the blood coagulation cascade and cellular components, notably platelets, which make up a large percentage of arterial thrombi. There is currently no clinically approved effective therapy for the treatment or prevention of acute arterial thrombosis or rethrombosis since heparin, the most widely used clinical anticoagulant administered i.v., has not been shown to be universally effective in this setting. Prins, M. H. and Hirsh, J., J. Am. Coll. Cardiol., 67:3A (1991).
Besides the unpredictable, recurrent thrombotic reocclusion which frequently occurs following PTCA, a profound restenosis of the recanalized vessel occurs in 30 to 40% of patients 1 to 6 months following this procedure. Califf, R. M. et al., J. Am. Coll. Cardiol., 17:2B (1991). Many of these patients require further treatment with either a repeat PTCA or coronary artery bypass surgery to relieve the newly formed stenosis which results in restriction of blood supply to the myocardium. Restenosis of a mechanically damaged vessel is not the direct result of a thrombotic process but instead is the result of a proliferative response of the vascular smooth muscle cells constituting the wall of the artery. Over time this results in a decreased luminal diameter of the affected vessel and decreased blood flow due to increased cellular and pericellular mass. Id. As for arterial thrombosis, there is currently no effective pharmacologic treatment for the prevention of vascular restenosis following mechanical recanalization.
The need for safe and effective therapeutic anticoagulants has in one aspect focused on the role of thrombin as the final enzyme in the process of blood coagulation.
As previously mentioned, recurrent arterial thrombosis remains one of the leading causes of failure following enzymatic or mechanical recanalization of occluded coronary vessels using thrombolytic agents or percutaneous transluminal coronary angioplasty (PTCA), respectively. After lysis of a clot by enzymatic means, residual thrombi may be responsible for reocclusion of the recanalized coronary artery via increased thrombus growth. Gash, A. K. et al., Am. J. Cardiol., 57:175 (1986); Shaer, D. H. et al., Circulation, 76:57 (1984). Mechanical recanalization by coronary angioplasty may not prevent reocclusion, and in the presence of a residual thrombus, may precipitate acute reocclusion, requiring bypass surgery. Sugrue, D. et al., Br. Heart J., 56:62 (1986). The development of methods for direct thrombus imaging have been stimulated by these clinical problems.
In vivo diagnostic imaging for intravascular thrombi has been reported. These imaging methods use compounds which are detectable by virtue of being labelled with radioactive or paramagnetic atoms. For example, platelets labelled with the gamma emitter, In-111, have been reported as an imaging agent for detecting thrombi. Thakur, M. L. et al., Thromb. Res., 9:345 (1976); Powers et al., Neurology, 32:938 (1982). A thrombolytic enzyme, such as streptokinase, labelled with the gamma emitter Tc-99m, has been proposed as an imaging agent. Wong, D. W., U.S. Pat. No. 4,418,052 (1983). The fibrin-binding domains of Staphylcoccus aureus derived protein A labelled with the gamma emitters, 1-125 and 1-131, have been proposed as imaging agents. Pang, R. H. L., U.S. Pat. No. 5,011,686 (1991). Monoclonal antibodies having specificity for fibrin (in contrast to fibrinogen) and labelled with the gamma emitter, Tc-99m, have been proposed as imaging agents. Berger, H. J. et al., U.S. Pat. No. 5,024,829 (1991); Dean, R. T. et al., U.S. Pat. No. 4,980,148 (1990). The use of the paramagnetic contrasting agent, gadolinium diethylenetriaminepentaacetic acid, in magnetic resonance imaging of patentis treated by thrombolysis for acute myocardial infarction has been reported. De Roos, A. et al., Int. J. Card. Imaging, 7:133 (1991).
Most preferred natural substrates for thrombin are reported to contain an uncharged amino acid in the P3 recognition subsite. For example, the thrombin cleavage site on the A.alpha. chain of fibrinogen, which is a physiological substrate for thrombin, is reported to contain a glycine residue in this position while the cleavage site on the B.beta. chain contains a serine, as shown below:
P4 P3 P2 P1 P1'
Gly--Gly--Val--Arg/Gly Fibrinogen A.alpha. Chain [SEQ. ID. NO. 1]
Phe--Ser--Ala--Arg/Gly Fibrinogen B.beta. Chain [SEQ. ID. NO. 2]
Peptidyl derivatives having an uncharged residue in the P3 position which are believed to bind to the active site of thrombin and thereby inhibit the conversion of fibrinogen to fibrin and cellular activation have been reported. Additionally, these derivatives have either an aldehyde, chloromethyl ketone or boronic acid functionality associated with the P1 amino acid. For example, substrate-like peptidyl derivatives such as D-phenylalanyl-prolyl-argininal (D--Phe--Pro--Arg--al), D-phenylalanyl-prolyl-arginine-chloromethyl ketone (P-PACK) and acetyl-D-phenylalanyl-prolyl-boroarginine (Ac--(D--Phe)--Pro--boroArg) have been reported to inhibit thrombin by directly binding to the active site of the enzyme. Bajusz, S., Symposia Biologica Hungarica, 25:277 (1984), Bajusz, S. et al, J. Med. Chem., 33:1729 (1990) and Bajusz, S. et al., Int. J. Peptide Protein Res. 12:217 (1970); Kettner, C. and Shaw, E., Methods Enzymol., 80:826 (1987); Kettner, C. et al., EP 293,881 (published Dec. 7, 1988); Kettner, C., et al., J. Biol. Chem., 265:18209 (1990). These molecules have been reported to be potent anticoagulants in the prevention of platelet-rich arterial thrombosis. Kelly, A. B. et al., Thromb. Haemostas., 65:736 at abstract 257 (1991).
Peptidyl compounds which are said to be active site inhibitors of thrombin but which are said to differ in structure from those containing a uncharged amino acid in the P3 recognition subsite have been reported. The compound, Argatroban (also called 2R, 4R-4-methyl-1-[N-2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arg ininal]-2-piperdinecarboxylic acid), is also reported to bind directly to the active site of thrombin and has been thought to be the most potent and selective compound in the class of non-peptidyl inhibitors of this enzyme. Okamoto, S. et al., Biochem. Biophys. Res. Commun., 101:440 (1981). Argatroban has been reported to be a potent antithrombotic agent in several experimental models of acute arterial thrombosis. Jang, I. K. et al., in both Circulation, 81:219 (1990) and Circ. Res., 67:1552 (1990).
Peptidyl compounds which are said to be inhibitors of thrombin and whose mode of action is thought to be by binding to the active site as well as an accessory or exo-site on the enzyme have been reported. For example, hirudin and its various peptidyl derivatives have been reported to inhibit both conversion of fibrinogen to fibrin and platelet activation by binding to either both the active site and exo-site, or to the exo-site only, of thrombin. Markwardt, F., Thromb. Haemostas., 66:141 (1991). Hirudin is said to be one of the most potent inhibitors of thrombin known. Marki, W. E. and Wallis, R. B., Thromb. Haemostas., 64:344 (1990). Hirudin is reported to inhibit thrombin by binding to both its anion-binding exo-site and to its catalytic active site, sites which are distinct and physically distant from each other. Rydel, T. J. et al., Science, 249:277 (1990). Its potency as measured by the inhibitory constant ("Ki") was determined to be 22.times.10.sup.-15 M. Stone et al., Biochemistry, 25:4622, 4624 (1986). Hirudin has been reported to be a potent antithrombotic agent in vitro and in vivo. Markwardt, F. et al., Pharmazie, 43:202 (1988); Kelly, A. B. et al., Blood, 77:1 (1991). In addition to its antithrombotic effects, hirudin has been reported to also inhibit smooth muscle proliferation and the associated restenosis following mechanical damage to a atherosclerotic rabbit femoral artery. Sarembock, I. J. et al., Circulation, 84:232 (1991).
Hirudin has been reported to be a 65 amino acid polypeptide which was originally isolated from leech salivary gland extracts. The primary amino acid sequence, as shown below, has been reported. Krstenansky J. L. et al., Thromb. Hemostasis, 63:208 (1990). ##STR1## The primary amino acid sequence of various isoforms of hirudin has also been reported. Scharf M. et al., FEBS Lett., 255:105 (1989). The C-terminal portion (comprised of amino acids 56 to 64) of hirudin has been reported to be the minimal domain required for the binding of hirudin to the exo-site of thrombin. Krstenansky, J. L., et al., Thromb. Hemostasis, 63:208 (1990); Mao, S. J. T., et al., Biochemistry, 27:8170 (1988); Krstenansky, et al., FEBS Lett., 211:10 (1987). Peptides similar to this C-terminal portion have been reported to inhibit thrombin-induced clot formation and/or thrombin-mediated platelet aggregation.
Hirugen has been reported to be a peptide derived from the anionic carboxy-terminus of hirudin. It is reported to bind only to the anion binding exo-site of thrombin and thereby inhibit the formation of fibrin but not the catalytic turnover of small synthetic substrates which have access to the unblocked active site of the enzyme. Maraganore, J. M. et al., J. Biol. Chem., 264:8692 (1989). The region of hirudin represented by hirugen has been shown using x-ray crystallographic techniques to bind directly to the exo-site of thrombin. Skrzypczak-Jankun, E. et al., Thromb. Haemostas., 65:830 at abstract 507 (1991). Moreover, the binding of hirugen has also been reported to enhance the catalytic turnover of certain small synthetic substrates by thrombin, indicating that a conformational change in the enzyme active site may accompany occupancy of the exosite. Naski, M. C. et al., J. Biol. Chem., 265:13484 (1990); Liu, L. W. et al., J. Biol. Chem., 266:16977 (1991). Hirugen also is reported to block thrombin-mediated platelet aggregation. Jakubowski, J. A. and Maraganore, J. M., Blood, 75:399 (1990). The inhibition of thrombin-induced fibrin clot formation resulting from substitution of the various amino acid residues on a C-terminal peptide of hirudin has also been reported. Krstenansky, J. L., et al., Thromb. Hemostasis, 63:208 (1990).
A chimeric peptide has been reported to be comprised of a C-terminal peptide of hirudin (amino acids 53 to 64) coupled to a peptide containing an Arg--Gly--Asp (RGD) sequence. The C-terminal peptide, with or without the RGD-containing peptide, is said to inhibit both thrombin-induced clot formation and thrombin-mediated platelet aggregation with an IC.sub.50 of 0.6 .mu.M and 7 .mu.M, respectively. Church, F. C. et al., J. Biol. Chem., 266:11975 (1991).
Another chimeric peptide, Hirulog, has been reported to be a synthetic molecule comprised of a hirugen-like sequence (amino acids 53 to 64 of hirudin) linked by a glycine-spacer region to the peptide, D-phenylalanyl-prolyl-arginine. The latter portion of this peptide is said to be based on a preferred substrate recognition site for thrombin. The hirugen-like sequence is said to be located at the C-terminus of this peptide. Maraganone, J. M. et al., Biochemistry, 29:7095 (1990); Maraganone, J. M. et al., International Application No. WO 91/02750 (published Mar. 7, 1991); and Dimaio, J. et al., International Application No. WO 91/19734 (published Dec. 26, 1991). Hirulog is said to bind to thrombin in a bivalent manner and this binding is characterized by an Ki of 2.56.times.10.sup.-9 M. The D-phenylalanyl-prolyl-arginine peptide is said to bind to the catalytic site of thrombin, whereas the hirugen-like sequence binds to its anion-binding exo-site. Witting, J. I. et al., Biochem. J., 283:737 (1992). Hirulog has been reported to be an effective antithrombotic agent in vivo, preventing both fibrin-rich and platelet-rich thrombosis. Maraganone, J. M. et al., Thromb. Haemostas., 65:651 at abstract 17 (1991).
Hirulog has been reported to have the structure, H--(D--Phe)--Pro--Arg--Pro--(Gly).sub.4 -Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu, and is said to be potent thrombin inhibitor. The substitution of various amino acids on the hirugen-like sequence of Hirulog and the effect thereof on binding constant has been reported. Bourdon, P. et al., FEBS, 294:163 (1991). Substitution of the D-phenylalanine residue with a .beta.-cyclohexyl-D-alanine residue is said to provide a more potent thrombin inhibitor, characterized by a Ki of 0.077.times.10.sup.-9 M. Witting, J. I. et al., Biochem. J., 287:663, 664 (1992). Addition of a methylene group between the arginine .alpha.-carbon and carbonyl of Hirulog is said to provide a non-clearable thrombin inhibitor characterized by a Ki of 7.4.times.10.sup.-9 M, while substitution of a methylene group for this carbonyl alone is said to provide a poor thrombin inhibitor having a Ki of greater than 2000.times.10.sup.-9 M. Kline, T. et al., Biochem. Biophys. Res. Commun., 177:1049, 1052-1054 (1991). N-acetyl--D--Phe--Pro Arg--[.psi.C(.dbd.O)--CH.sub.2 ]--CH.sub.2 --CH.sub.2 --CH.sub.2 -(C.dbd.O)--Gln--Ser--His--Asn--Asp--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Gl u--Glu--Tyr--Leu--Gln is said to be potent thrombin non-cleavable inhibitor having a Ki of 0.14.times.10.sup.-9 M. Dimaio, J. et al., International Application, at page 44.
Cyclotheonamide A and B, isolated from the marine sponge, Theonella, a genus of marine sponges, have been reported to be inhibitors of thrombin with an IC.sub.50 of 0.076 .mu.g/mL (9.9.times.10.sup.-8 M). Structurally, they have been characterized as cyclic peptides containing an arginine .alpha.-keto amide moiety. Fusetani et al., J. Am. Chem. Soc. 112:7053-7054 (1991) and Hagihara et al., J. Am. Chem. Soc, 114:6570-6571 (1992). It has been proposed that the .alpha.-keto group of the cyclotheonamides may function as an electrophilic mimic of the Arg--X scissile amide bond of the thrombin substrates. Hagihara et al., Id. at 6570. The partial synthesis of gyclotheonamide A and the total synthesis of cyclotheonamide B have been reported. Wipf et al., Tetrahedron Lett., 33:4275-4278 (1992) and Hagihara et al., J. Am. Chem. Soc, 114:6570-6571 (1992).
.alpha.-Keto-amide derivatives of other amino acids and peptides have also been reported to be inhibitors of proteases. For example, L--valyl--L--valyl--3-amino-2-oxovaleryl-D-leucyl-L-valine had been reported to be an inhibitor of prolyl endopeptidase. Nagai et al., J. Antibiotics, 44:956-961 (1991). 3-Amino-2-oxo-4-phenylbutanoic acid amide has been reported to be an inhibitor of arginyl aminopeptidase (with a Ki of 1.5 .mu.M), cytosol aminopeptidase (with a Ki of 1.0 .mu.M) and microsomal aminopeptidase (with a Ki of 2.5 .mu.M). Ocain et al., J. Med. Chem., 35:451-456 (1992). 2-Oxo-2-(pyrrolidin-2-yl) acetyl derivatives have been reported to be inhibitors of prolyl endopeptidase. Someno et al., European Patent Application No. 468,339 (published Jan. 29, 1992). Certain .alpha.-keto-amide derivatives of peptides have been reported to inhibit various serine and cysteine proteases. Powers J. C., International Application No. WO 92/12140 (published Jul. 23, 1992).
.alpha.-Keto ester derivatives of N-protected amino acids and peptides have also been reported as inhibitors of serine proteases, such as neutrophil elastase and cathepsin. G. Mehdi et al., Blochem. Biophys. Res. Commun., 166:595-600 (1990) and Angelastro et al., J. Med. Chem., 33:11-13 (1990).